Modulation of intein activity by its neighboring extein substrates

Abstract

Inteins comprise a large family of phylogenetically widespread self-splicing protein catalysts that colonize diverse host proteins. The evolutionary and functional relationship between the intein and the split-host protein, the exteins, is largely unknown. To probe an association, we developed an in vivo and in vitro intein assay based on FRET. The FRET assay reports cleavage of the intein from its N-terminal extein. Applying this assay to randomized extein libraries, we show that the nature of the extein substrate bordering the intein can profoundly influence intein activity. Residues proximal to the intein-splicing junction in both N- and C-terminal exteins can accelerate the N-terminal cleavage rate by >4-fold or attenuate cleavage by 1,000-fold, both resulting in compromised self-splicing efficiency. The existence and the magnitude of extein effects require consideration for maximizing the utility of inteins in biotechnological applications, and they predict biases in intein integration sites in nature.

Fig. 1.

FRET-based N-extein cleavage assay. (A) FRET reporter construct. Splicing and N-extein cleavage pathways for a FRET-active intein precursor, CFP-Intein-YFP (C-I-Y), and relative FRET associated with the respective reaction products are shown. (B) Intein–extein structure relationship. Ssp PCC6803 DnaE intein (red) with its native N-2, N-1 (blue), C+2, and C+3 (yellow) extein residues, and catalytic residues Cys-1 and Asn-159, and Cys+1 residues (green) (modified from PDB:1ZD7).

Fig. 2.

FRET assay reports N-extein cleavage in vitro. (A) Progress of N-extein cleavage. Recorded FRET values over 1 h with C-Ia-Y extracts and controls (C-Iaa-Y; C+IY, mixture of C and I-Y, purified separately) in the presence and absence of DTT (20 mM). (B) Mechanism of DTT-induced N-extein cleavage. (C) Gel analysis of N-extein cleavage. Unheated extracts of C-Ia-Y with (+) or without (−) DTT (20 mM) separated by 12% SDS/PAGE followed by fluorescence detection of YFP and CFP (excitation 457 nm, emission 526 nm; wavelengths overlap for CFP and YFP). Lane 1: BG, blank MC1061 lysate. Both bands in the doublet labeled “C” are CFP (lane 7), which migrates spuriously in native form on an SDS gel.

Fig. 3.

FRET assay for in vivo screening and in vitro characterization of cleavage mutants. (A) Representative FRET screening of an extein mutant library. Dual emission plot of data collected from a live-cell expression library of the C-Ia-Y variants with randomly mutated N-2 and N-1 extein residues. (Inset) FRET measured in vivo for C-Iaa-Y, C-Ia-Y, and n-ND 6 h postinduction, relative to E. coli MC1061 (BG). (B) Extein effects on DTT-induced N-extein cleavage. Progress of N-extein cleavage of parental C-Ia-Y along with representative extein mutants that show suppressed (red) and accelerated (green) reactivity toward DTT in vitro. Mutants of intermediate phentoype are shown in black. Identified variants were further characterized. (C) Rates of DTT-induced N-extein cleavage for 30 variants with k_obs values that differ by >2-fold from the parental C-Ia-Y, representing 4–5% of total variants screened. Residue combination of library n refers to randomized N-2 N-1 positions, library c to C+2 C+3, and library nc to N-2 N-1 and C+2 C+3. Bold letters designate variants that were further characterized.

Fig. 4.

Characterization of accelerators and attenuators in vitro. (A) Progress of DTT-induced N-extein cleavage. Soluble protein extracts of parental construct (C-Ia-Y), accelerator mutants n-ND and c-SC, and attenuator n-RP were incubated at pH 8 with 20 mM DTT for the times indicated. Reaction mixtures were separated by12% SDS/PAGE followed by fluorescence detection of YFP and CFP (excitation 457 nm, emission 526 nm for both CFP and YFP). (B) Single extein residue effects on N-terminal cleavage. Individual residues from 3 mutant extein pairs that produced accelerated (n-ND, c-SC; lanes 2 and 8) or reduced (n-RP; lane 5) rates of N-extein cleavage compared with native extein parental C-Ia-Y control (P, lane 1) were returned to their native congener residue (underlined). The spurious migration of CFP (C) is as in Fig. 2C. The percentage of precursor was determined by dividing the fluorescent intensity of the C-I-Y band by that of the I-Y+C-I-Y bands (excitation 488 nm, emission 580 nm). (C) Attenuated and induced N-extein cleavage with N-1 Pro. Spontaneous and DTT-induced N-extein cleavage for parental extein control (C-Ia-Y) and N-1 Pro mutant, n-RP, was determined after 18 h incubation. Protein extracts were incubated at pH 8 without (−) or with (+) 20 mM DTT (lanes 1–4) or without (−) and with (+) 0.1 M hydroxylamine (lanes 5–8), and the products separated by 12% SDS/PAGE followed by fluorescent gel imaging as above.

Fig. 5.

Divergent paths for N-extein cleavage. (A) Cleavage pathway in N159A (C-Ia-Y) mutant. Steps 1 and 2 are described in Divergent Pathways for N-terminal Cleavage. (B) The extents of in vivo N-extein cleavage. The n-ND and c-SC derivatives and their Ala point mutants at the catalytic intein Cys (C1) or extein Cys (C + 1) residues were analyzed alongside parental C-Ia-Y (P) as in Fig. 4. The ≈10-fold difference between the percentage of precursor in lanes 6 and 8 was reproducible over 3 independent experiments.

Fig. 6.

Splicing is altered by N- and C-extein residues. (A) Complete intein splicing pathway (described in Extein Effects on Splicing in Addition to Cleavage). (B) Commassie-stained gel of Ni²⁺-NTA-purified products from cultures expressing splicing competent C-I-Y (WT) and its extein mutants, along with Ni²⁺-NTA purified MC1061 lysate (BG). (C) Data summary. The relative abundance of precursor (C-I-Y), spliced product (C-Y), and N-terminal cleavage product (I-Y) apparent from B.

Scheme 1.